Chronic osteomyelitis risk is associated with NLRP3 gene rs10754558 polymorphism in a Chinese Han Population

Background Single nucleotide polymorphisms (SNPs) in the nucleotide-binding domain leucine-rich repeat protein-3 (NLRP3) gene are reported to be linked to many inflammatory disorders. However, uncertainty persists over the associations between these SNPs and susceptibilities to chronic osteomyelitis (COM). This study aimed to investigate potential relationships between NLRP3 gene SNPs and the risks of developing COM in a Chinese Han cohort. Methods The four tag SNPs of the NLRP3 gene were genotyped in a total of 428 COM patients and 368 healthy controlsusing the SNapShot technique. The genotype distribution, mutant allele frequency, and the four genetic models (dominant, recessive, homozygous, and heterozygous) of the four SNPs were compared between the two groups. Results A significant association was found between rs10754558 polymorphism and the probability of COM occurence by the heterozygous model (P = 0.037, odds ratio [OR] = 1.541, 95% confidence interval [CI] = 1.025–2.319), indicating that rs10754558 may be associated with a higher risk of developing COM.In addition, possible relationship was found between rs7525979 polymorphism and the risk of COM development by the outcomes of homozygous (P = 0.073, OR = 0.453, 95% CI = 0.187–1.097) and recessive (P = 0.093, OR = 0.478, 95% CI = 0.198–1.151) models, though no statistical differences were obtained. Conclusions Outcomes of the present study showed, for the first time, that rs10754558 polymorphism of the NLRP3 gene may increase the risk of COM development in this Chinese Han population, with genotype CG as a risk factor. Nonetheless, this conclusion requires verification from further studies with a larger sample size. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-024-01799-6.


Background
Chronic osteomyelitis (COM) is a frequent and challenging complication of trauma, affecting up to one third of patients during recovery from severe limb injury or open fracture [1,2].In addition, hematogenous seeding and soft tissue infections, especially in patients with diabetes and pressure ulcers, can also lead to osteomyelitis (OM) [3].Staphylococcus aureus remains to be the primary pathogen of COM [4,5].Owing to insufficient blood supply to the infected bone, patients with COM often require both surgical debridement and antibiotic treatment [6,7].COM has been shown to substantially increase the long-term mortality risk [8].Approximately 30% of the acute OM cases progress into chronic phase [9], causing anincreased risk of mortality, perpetuating disability, and worsened quality of life [8,10,11].Previous epidemiological studies have reported that COM elevates the risk of coronary heart disease [12], stroke [13], diabetes mellitus [14], renal disease [15], and even depression [10].To comprehensively tackle COM-related issues, substantial efforts should be devoted to investigate the pathophysiology of the condition as well as to conduct clinical studies.The pathogenesis of OM is related to both environmental factors and genetic factors, and contemporary evidence suggests that genetic susceptibility may also play a crucial role in this process [16,17].With the rapid development and application of sequencing and genetic association analysis, genetic variants that may cause OM have been extensively explored.
Numerous genetic studies examining nucleotide-binding domain leucine-rich repeat protein-3 (NLRP3) polymorphisms have demonstrated the involvement of this gene in a range of inflammatory conditions, including Parkinson's and Alzheimer's diseases [18], type 2 diabetes, atherosclerosis [19], inflammatory bowel disease [20], gout, and recurrent fever [21].Genetic differences in the NLRP3 have been hypothesized to have a substantial role in determining the severity of inflammatory responses, thereby predisposing susceptibility to disease [22].However, the association between NLRP3 and COM, as well as the underlying mechanisms remain unclear.Previous studies had reported that several single nucleotide polymorphisms (SNPs), such as TaqI (rs731236) and FokI (rs2228570) in the vitamin D receptor (VDR) gene [23], rs689466 in the cyclooxygenase-2 (COX-2) gene [24], the Alu insertion/deletion (rs4646972) in the tissue plasminogen activator (tPA) gene [25], and rs1799750 in the matrix metalloprotease 1 (MMP-1) gene [26], may be associated with the risk of developing COM.In the HaploReg v4.2-Broad Institute (HaploReg v4.2 (broadinstitute.org)),we found that rs10754558, rs7525979, rs35829419, and rs4612666 were all involved in inflammatory responses and all played key roles in the mouse OM model (Supplementary Table S1).
To better understand potential role of genetic factor in the pathogenesis of COM, this study examined potential links between NLRP3 SNPs and susceptibilities to COM in a Chinese Han population.

Study design and patient enrollment
This investigation was designed as a case-control study and conducted in a Chinese Han population.Patients undergoing treatment for COM between 2016 and 2019 at the Nanfang Hospital, Southern Medical University, a tertiary healthcare facility in Southern China, were screened for enrollment.COM was defined by symptoms persisting for at least 10 weeks and/or radiological appearances suggestive signs of bone infection.In addition, at least one of the following operative findings was required to be present for inclusion: (i) two or more positive culture sterile site specimens with an indistinguishable organism; (ii) histology suggestive of COM (a mean of > 5 neutrophils per high power field, averaged over at least 5 fields); or (iii) sinus abscess or purulence, present during surgery.These criteria align with established methods of confirming the diagnosis of COM.Healthy controls were individuals without abnormalities or a history of any disorders, as determined by thorough examinations in the physical examination center.Informed consent forms were completed by all the participants or their guardians, and the medical ethics committee of the Southern Medical University Nanfang Hospital approved the study (approval no.NFEC-2019-087).

SNP selection and genotyping
Data regarding NLRP3 gene expression in osteoblasts were obtained through gene sequencing (BioProject number: PRJNA1001643).The model included a normal osteogenic differentiation group (tumor necrosis factor [TNF-α] = 0 ng/mL) and an inflammasome group (TNFα = 5 ng/mL).The expression of the NLRP3 gene was then compared between the samples belonging to these two groups.We analyzed four selected SNPs (rs35829419, rs10754558, rs7525979, and rs4612666) in the NLRP3 inflammasome gene.Previous studies on inflammasome gene polymorphisms related to infection were referred.The expressions of these four SNPs in tissues were verified in the Genotype-Tissue Expression (GTEx) database.The GTEx project is an ongoing effort to build a comprehensive public resource to study tissue-specific gene expression and regulation.It provides open access to information, including gene expression, quantitative trait loci data, and histology images.
Using the Multiplex SNaPshot technique, the four SNPs of the NLRP3 gene were genotyped.SNaPshot extension reactions were performed following the instructions of the ABI SNaPshot Multiplex PCR Kit (Applied Biosystems, Waltham, MA, USA) with slight revisions.The total reaction volume (6.0 µL) included 2.0 µL of purified PCR products, 1.0 µL of SNaPshot Multiplex Ready Reaction Mix, 0.2 µL of 10 µmol/mL pooled extension primer, and 2.8 µL of ddH 2 O. Extension reactions were performed as follows: pre-denaturation at 96 °C for 1 min, followed by 30 cycles of denaturation at 96 °C for 10 s, annealing at 52 °C for 5 s, and extension at 60 °C for 30 s.The product of this reaction (1.0 µL) was mixed with 9.0 µL of formamide and denatured at 95 °C for 3 min.The fluorescently labeled fragments were separated by capillary electrophoresis on an ABI PRISM 3730 XL Genetic Analyzer (Applied Biosystems).The results of the SNaPshot genotyping method are shown in Fig. 1.

Comparative analysis of NLRP3 gene polymorphisms and clinical indicators
Genotype distribution, mutant allele frequency, and the four genetic models (dominant, recessive, homozygous, and heterozygous) of the four NLRP3 SNPs were compared between the COM patients and healthy controls.Age, sex ratio, culture-positive rate, polymicrobial infection rate, and preoperative serum levels of two inflammatory biomarkers, interleukin (IL)-6 and TNF-α, were compared between the four genotypes of NLRP3 gene polymorphisms among the patient group.Serum levels of IL-6 and TNF-α were detected using electrochemiluminescence immunoassay (Roche cobas e601, Basel, Switzerland).The upper limit of normal values, provided by the Medical Clinical Laboratory of Nanfang Hospital, were 7.0 and 8.1 pg/mL for IL-6 and TNF-α, respectively.

Statistical analysis
The Kolmogorov-Smirnov test assessed data normality.Normally distributed continuous variables are reported as the mean ± standard deviation, and a one-way analysis of variance or the student's t-test was used to compare between the two or among over two groups.Continuous variables with a non-normal distribution were reported as the median withinterquartile range (IQR), and the Kruskal-Wallis or Mann-Whitney U tests were used for group comparisons.Dichotomous variables with percentage-based data were compared using the Chi-square or the Fisher's exact test.
Analysis of the linkage disequilibrium at different loci of NLRP3 genotypes was performed.The genotype distributions of the healthy controls were examined using the Chi-square test to determine whether Hardy-Weinberg equilibrium (HWE) were sustained.The Chi-square test or Fisher's exact test was used to examine the genotype distributions and mutant allele frequencies between the two groups.Sex, age, and genotype distribution were covariates.Binary logistic regression analysis was performed to examine the potential relationships between the four gene polymorphisms and the likelihood of developing COM using four genetic models with matching odds ratios (ORs) and 95% confidence intervals (CI).Statistical analyses were performed using R software (Version 4.2.3,LD-heatmap package) and SPSS version 25.0 (IBM Corp., Armonk, NY, USA).Statistical significance was established as a P-value < 0.05.

Clinical characteristics of the participants
This study included 368 healthy controls (268 men and 100 women) and 428 COM patients (337 men and 91 Table 1 Forward, reverse, and extension primers of the 4 tag SNPs for PCR

5'-T T C T C C A A G C T C C C A C C A A T A C T A C-3'
SNP: Single Nucleotide Polymorphism; PCR: Polymerase Chain Reaction; F: Forward; R: Reverse women).There was no statistically significant difference in the sex ratio between the two groups (3.70 vs. 2.68, χ 2 = 3.792, P = 0.051).The median ages of the two groups were not significantly different (patient group: 47 years, IQR, 33.00-59.00;control group: 46 years, IQR, 37.00-52.00;Z = 0.114, P = 0.662).Figure 2 shows clinical characteristics of COM of this Chinese cohort.The most prevalent type of COM was post-traumatic OM (PTOM)(58.35%),which often occurred after open injury (61.96%).The most typical infection location was the tibia (38.31%), followed by the foot (21.03%) and the femur (11.68%).Of all the intraoperative specimen cultures, 27.46% were tested positive, with monomicrobial infection accounting for 71.67%.The most commonly detected pathogen was Staphylococcus aureus (34.17%).

Expression of NLRP3 in different states and linkage disequilibrium analysis
The expression of the NLRP3 gene under inflammatory microenvironment was significantly higher than that  S2) under normal microenvironment (P = 0.015) (Fig. 3).However, according to the GTEx database, the expression of NLRP3 gene variants (rs10754558, rs7525979, rs35829419, and rs4612666) in the whole-blood and the musculoskeletal tissues did not show significant differences.The r 2 values of the NLRP3 gene SNPs were calculated using R software (Version 4.2.3).The results of the linkage disequilibrium analysis were shown in the r 2 hot graph in Fig. 4. Deep-blue blocks represent high linkage disequilibrium (r 2 = 0.8-1), and light-blue blocks represent low linkage disequilibrium (r 2 = 0-0.2).There was no linkage disequilibrium among the three SNPs of NLRP3 (rs10754558, rs7525979, and rs35829419).

Frequency of the four NLRP3 gene SNPs in COM patients and healthy controls
In the healthy controls, all the four genotyped NLRP3 gene SNPs were in HWE: P (HWE) for rs35829419 = 0.979, P (HWE) for rs10754558 = 0.377, P (HWE) for rs7525979 = 0.386, and P (HWE) for rs4612666 = 0.607.As shown in Table 2, the heterozygous model of the rs10754558 SNP and susceptibility to COM were significantly associated (P = 0.037, OR = 1.541, 95% CI = 1.025-2.319),indicating that individuals with the CG genotype may be at a higher risk in developing COMin this Chinese Han population.Although no significant association was identified between rs7525979 and the risk of developing COM, results of the recessive (P = 0.093) and homozygous (P = 0.073) models suggested that this SNP site may be linked to a decreased risk of COM development, with genotype TT as a possibly protective factor.However, this results need to be further tested.No significant correlations were found between rs35829419 or rs4612666 and the risks of COM development among these participants (Table 2).

IL-6 and TNF-α levels among different genotypes of rs10754558 and rs7525979 in the COM patients
Serum levels of IL-6 and TNF-α in the COM patients showed no significant differences among different genotypes of rs10754558 (P = 0.213 and 0.662, respectively; Table 3).Additionally, multiple comparisons showed that no significant differences were found regarding serological IL-6 or TNF-α levels between CG and GG (P = 0.180), CG and CC (P = 0.158), or CG and CC + GG (P = 0.072) genotype groups (Fig. 5).Moreover, serum levels of IL-6 and TNF-α did not differ significantlyamong different genotypes of rs7525979 (P = 0.900 and 0.843, respectively; Table 3) (Fig. 6).

Discussion
DOur study results showed that, in this ChineseHan cohort, NLRP3 SNP rs1074558 may increase the risk of COM development, with CG genotype in a higher risk to develop COM.Despite the current study being unable to obtain statistical evidence supporting this association between the COM patients and healthy controls, the results of the recessive and homozygous models suggested that rs7525979 polymorphism may hinder COM development.Nonetheless, this conclusion requires verification from further studies with a larger sample size.
COM, which often develops following trauma and orthopaedic surgery, still poses great challenges tophysicians.The frequency of infection in the long bones after open fractures varies between 4% and 64%, with infection recurrence rates ranged between 20% and 30% [27].Among the 428 COM patientsincluded in our study, PTOMaccounted for 58.35%, with infection following open injury occupying 61.96% and the positive rate of culture as27.46%.The physical and psychological wellbeing of patients is severely affected by the protracted course of the disease and inflammatory bone disintegration.Huang et al. [28] found that COM significantly increased the risk of death in the older population.Along with the aforementioned comorbidities, patients with COM also suffer fromhigher rates of deep vein thrombosis [15], erectile dysfunction [29], dementia [30], and end-stage renal disease [31], as well as mental health disorders, including depression [10].In addition, while rare, there is a risk of malignant change in patients with COM [32].Consequently, COM is accompanied by an enormous healthcare and economic burdens [33].
Previous studies have found that IL-1β secretion and mRNA expression levels of key inflammasome components, namely apoptosis-associated speck-like protein containing a CARD and Caspase-1, are abnormally increased in patients with chronic recurrent multifocal osteomyelitis [34,35].The release of IL-1β can lead to autocrine stimulation and production of other cytokines, such as IL-6.In the present study, we did not detect the serological level of IL-1β.Thus, the potential influences of such SNPs on serological levels of IL-1β cannot be assessed.Instead, we evaluated the impact of genotype by comparing readily available serum inflammatory markers, IL-6 and TNF-α.
Studies have shown that genetic variables, with SNP as a key component, may play a role in the etiology of infection.According to several clinical studies, the VDR [23], tPA [25], COX-2 [24], and MMP-1 [26] genes, and the most frequently reported members of the IL family, have been implicated in the associations between SNPs and COM risk across various ethnicities.Previous investigations reported that IL-1 (rs1800587) [36,37], IL-4 (rs2243250, rs2243248) [37], and IL-6 (rs1800795) [37] gene polymorphisms are positively correlated with the development of COM and raise the risk of developing OM.And we found that these SNPs pathways are associated with inflammation.As a crucial component of the inflammatory system, the NLRP3 inflammasome may Fig. 4 Linkage disequilibrium analysis.The r 2 hot graph for the single nucleotide polymorphisms of the nucleotide-binding domain and leucinerich repeat protein-3 gene: rs10754558, rs7525979, and rs35829419 also be linked to the incidence and progression of COM [38].More importantly, our team investigated a possible association between NLRP3 gene polymorphisms and the risks of developing PTOM in a Chinese population.The results suggested that NLRP3 SNPs, rs10754558 (P = 0.047) and rs7525979 (P = 0.048), were significantly different between patients and healthy controls.However, this study only discussed the susceptibility to PTOM and NLRP3 gene expressions, and could not clarify whether this gene susceptibility was related to COM.At the same time, we summarized the SNPs that are associated with COM, as well as the mechanistic pathways involved and the action (cis or trans) and location of SNPs (Table 4).Therefore, we conducted a more detailed study [39].
The rs10754558 polymorphism of the NLRP3 gene may be associated with an increased risk of COM in this Chinese Han population; to the best of our knowledge, no study has reported on this association to date.Although we did not find a difference in the frequency of the mutant alleles, C and G, of rs10754558 in the patient group, the heterozygous model of rs10754558 showed a statistical association of this polymorphism with COM, indicating that individuals with the CG genotype of this polymorphism are in an increased risk of COM development.Other genotypes did not significantly associated with COM.In addition, although there was no statisticalcorrelation between the results of the recessive and homozygous models of rs7525979 (P = 0.093 and 0.073, respectively), the frequency of the TT genotype was higher in the COM patients than that in healthy controls.Further research is required to verify whether patients with TT genotype of rs7525979 are in a lower risk to COM: Chronic osteomyelitis; OR: odds ratio, CI: confidence interval, NA: not available develop thisdisease.Several possible reasons may help explain why only the rs10754558 variants showed significant results while the others failed.The first reason may rest with the specificity of rs10754558 itself.Aside from the present findings, we also found that this SNP is associated with the development of several disorders, such as chronic kidney disease [40], acne vulgaris [41] and autoimmune diseases [42].Second, the limited sample size of our study may have masked the correlations between the other SNPs and COM susceptibilities.Therefore, we will explore whether and how these genetic variants play roles in epigenetics in the future.Moreover, no significant differences were found among different genotypes of rs10754558 or rs7525978 SNPs, neither regarding clinical characteristics, nor regarding serological levels of IL-6 or TNF-α.These results suggest that further studies are required to investigate the potential influences of NLRP3 polymorphisms on clinical features and serum levels of the inflammatory biomarkers as well.Nonetheless, outcomes of the present study also need to be confirmed by a larger cohort.
Our study had several limitations.Firstly, despite a larger sample size of the current report compared to the majority of the previously published studies, it remains inadequate for an SNP analysis.Additionally, a broader pool of participants should be applied in future research since the pathogenic process for the development of COM is multifactorial.Secondly, a few participants could not be genotyped using the SNapShot method.Owing to the limited volume of blood collection from each participant, genotyping could not be repeated using alternative methods, resulting in a reduced number of samples with sequencing outcomes.However, this did not affect the final results.Thirdly, we did not detect serum level of IL-1β, thus, IL-1β levels among patients with different genotypes could not be compared, and correlations between the NLRP3 gene polymorphisms and serum IL-1β levels remain unclear.Fourthly, the environmental variables may play a role in the pathophysiology of COM in addition to the genetic factors.The majority of the patients in this study were transferred from neighboring hospitals; thus, it was difficult to determine their precise medical history prior to bone infection (e.g., the severity of the original injury and treatment approach).As a result, we were unable to further investigate how these external factors affected the development of COM.Finally, we found that NLRP3 gene polymorphisms are associated with an increased risk of COM; however, the function of the genetic variants has not been analyzed.Therefore, further functional validations of NLRP3 gene polymorphisms are needed.

Conclusions
This study confirmed that NLRP3 gene rs10754558 polymorphism is significantly associated with the prevalence of COM in a Chinese Han population.Nonetheless, this conclusion requires validation by future studies with a larger sample size.Based on our findings, detecting overexpression of the NLRP3 mutant gene may be a promising strategy to identify risk group for COM development.
Future studies exploring this association that include a larger population of COM patients are essential, especially if genetic screening is to be employed as a prediction tool for the risk of disease development.Further, determining whether pharmacological inhibition of the NLRP3 pathway should be incorporated into the treatment regimen for COM may be beneficial.

Table 2
Comparisons of genotype distribution, allele frequency, and genetic models of rs10754558, rs7525979, rs35829419 and rs4612666 between the COM patients and healthy controls

Table 4
Previously reported SNP sites associating with OM development